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Published on: March 18, 2014
Deoxypodophyllotoxin Mediates Autophagy Death through Inhibition of GRP78 in Human Osteosarcoma
Xiao-Jun Tang1, Ling-Li Luo2, Wen-Chao Zhou3
1The Second Affiliated Hospital, Department of Spinal Surgery, Hengyang Medical School, University of South China, Hengyang, Hunan, 421001, China.
Background:
Glucose-regulated protein 78 (GRP78), as a chaperone protein, can protect the endoplasmic reticulum of cells and is expressed to influence chemoresistance and prognosis in cancer. Deoxypodophyllotoxin (DPT) is a compound with antitumor effects on cancers. DPT inhibits the proliferation of osteosarcoma by inducing apoptosis, necrosis, or cell cycle arrest.
Objectives:
This study was performed to demonstrate the molecular mechanism by which DPT attenuates osteosarcoma progression through GRP78.
Methods:
Natural compound libraries and western blot (WB) were used to screen the inhibitors of osteosarcoma GRP78. The expression of mitochondria-related genes in cancer cells of the treatment group was detected by quantitative real-time PCR (qPCR) and WB. 3-(4,5)- Dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT) and 5-ethynyl-2'- deoxyuridine (EDU) were used to discover the activity and proliferation of osteosarcoma cells treated with DPT. We constructed an in vivo mouse model of DPT drug therapy and carried out immunohistochemical detection of xenografts. The treated osteosarcoma cells were analyzed using bioinformatics and electron microscopy. The data were analyzed finally.
Results:
DPT inhibited osteosarcoma cell survival and the growth of tumor xenografts. It promoted up-regulation of BCL2-associated X protein (Bax) and B-cell CLL/lymphoma 2 (Bcl-2), which serves to mediate and attenuate, respectively, the killing activities of DPT through mitochondria dysfunction. The effect of DPT against cancer cells could be attenuated by the overexpression of GRP78, characterized by the inactivation of the caspase cascade. The loss of GRP78 in osteosarcoma cells negatively mediated the basal level of autophagyassociated genes. DPT stimulated autophagy via the phosphoinositide 3-kinase (PI3K)-v-akt murine thymoma viral oncogene homolog (AKT), a mechanistic target of rapamycin (mTOR) axis. The autophagy caused by DPT played an active role in the osteosarcoma of humans and blocked the apoptotic cascade.
Conclusion:
Combination treatment with the GRP78 inhibitor DPT and pharmacological autophagy inhibitors will be a meaningful method of obviating osteosarcoma cells.
Insights
Deoxypodophyllotoxin (DPT) inhibits osteosarcoma progression by targeting Glucose-regulated protein 78 (GRP78). This study reveals DPT stimulates autophagy, offering a potential therapeutic strategy for osteosarcoma treatment.
Area of Science:
- Oncology
- Molecular Biology
- Pharmacology
Background:
- Glucose-regulated protein 78 (GRP78) is a chaperone protein involved in cancer chemoresistance and prognosis.
- Deoxypodophyllotoxin (DPT) exhibits antitumor effects by inhibiting osteosarcoma cell proliferation through apoptosis, necrosis, or cell cycle arrest.
Purpose of the Study:
- To elucidate the molecular mechanism by which DPT attenuates osteosarcoma progression, focusing on the role of GRP78.
- To investigate the interplay between DPT, GRP78, and autophagy in osteosarcoma.
Main Methods:
- Screening of GRP78 inhibitors using natural compound libraries and Western blot (WB).
- Quantitative real-time PCR (qPCR) and WB to assess mitochondria-related gene expression.
- MTT and EDU assays for cell activity and proliferation; in vivo mouse models and immunohistochemistry for xenograft analysis.
Main Results:
- DPT inhibited osteosarcoma cell survival and tumor xenograft growth.
- DPT modulated mitochondria dysfunction by up-regulating Bax and down-regulating Bcl-2, while GRP78 overexpression attenuated DPT's effects by inhibiting the caspase cascade.
- DPT stimulated autophagy via the PI3K-AKT-mTOR pathway, which contributed to blocking the apoptotic cascade in osteosarcoma.
Conclusions:
- DPT's anti-osteosarcoma effects are mediated through GRP78 and involve the induction of autophagy.
- Combined inhibition of GRP78 and pharmacological autophagy inhibition presents a promising therapeutic approach for osteosarcoma.
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